The long-term goal of this research program is to elucidate the role of clathrin and the clathrin-coat structure in the membrane dynamics of cells. Coated membranes have been found in all nucleated eucaryotic organisms examined and clathrin, the major component of the coat structure, is a highly conserved protein. These observations, and the involvement of coated membranes in receptor-mediated endocytosis, secretion and membrane recycling, suggest that coat structures have a central role in membrane biology. To understand the functions of coated membranes at the molecular level, the mechanisms responsible for generating the coat structure at discrete sites will be studied. It is hypothesized that unique membrane attachment sites for clathrin exist on coated vesicles and this will be tested using a sedimentation assay and iodinated clathrin. The binding reaction will be characterized and the activity solubilized using detergents, chaotropes, proteases, etc. Purification of the factor will be monitored by its binding to clathrin using immunoprecipitation or competition with stripped vesicles as an assay. Reassembly of the coat structure from solubilized clathrin will be studied using electron microscopy, fluorescence polarization and energy transfer techniques as assays. The role of a previously identified assembly protein in this process will be characterized and the protein isolated. Monospecific anti-clathrin antibodies will be prepared and a radioimmunoassay for clathrin devised. With these antibodies, basic information about the distribution and turnover of clathrin and the intact coated vesicle will be obtained to determine if mechanisms exist to regulate clathrin levels in cells.